We are using matrix-assisted laser desorption ionization (MALDI) of peptides as a model system to study peptide ion fragmentation. Ion energetics relationships between laser fluence and peptide ion fragmentation. This type of study is fundamental to optimizing MALDI TOF/TOF experiments for the purpose of peptide sequencing. In these studies we obtain peptide fragmentation spectra, typically 5000 laser shots, in both the unimolecular decomposition and collision induced dissociation (CID) modes. We have the ability to easily follow two time points for each peptide decomposition, i.e., the in-source fragmentation consisting of ions formed within 1 usec after the laser firing and the longer, mass dependent fragmentation occurring within the instrument's collision cell. We have used the fragmentation of a model peptide, leucine enkephalin, YGGFL, (LeuEnk) over the full range of laser fluence as the basis of the initial studies. While not a peptide of the type normally encountered in protein characterizations, LeuEnk is an excellent model to enable studies of short lived processes in the laser plume. LeuEnk fragmentation spectra have been acquired in both MS and MS-MS using three different common matrices using a laser pulse length of 600 psec, compared to 5 nsec used previously. Spectra are acquired as a function of laser fluence beginning at the onset of ionization and extending to the maximum fluence available in the instrument. The spectra obtained using a-cyano-hydroxycinnamic acid (ACHA) reveal several distinct processes in LeuEnk fragmentation. First, the MS mode spectra show a region of extensive fragmentation occurring in what must be a very short time frame following the onset of ionization. These rapid fragmentations, leading to the observation only of immonium ions, are associated with the laser pulse-induced direct vaporization of molecules from the sample surface. A second set of process takes place within the first several hundred nanoseconds following the laser pulse. These processes, also manifest in MS mode, are most likely associated with desorption of LeuEnk ions from particles, consisting principally of matrix, ablated from the sample surface. These desorbed ions undergo a large number of collisions with the high temperature gases present in the laser plume, and begin to fragment; these fragmentations proceed in a series of consecutive reactions in which the amide backbone bonds are ruptured. Our spectra show that the initial direct desorption processes reach a maximum at about 50% of the total laser fluence, and then increase no further; at that point, the consecutive fragmentation reactions supplant them in intensity. Finally, the MS-MS mode spectra exhibit little fragmentation, most likely due to depletion of the high-energy portions of the energy distributions associated with the second stage, particle desorption processes, described above. Use of either 2,5-dihydroxy benzoic acid (DHB) or di-methoxy-hydroxy cinnamic acid (sinapinic acid, SA) leads to spectra with much less fragmentation than observed with ACHA coupled with a much higher fluence for the onset of the protonated LeuEnk molecule itself. in contrast to the extensie production of immonium ions observed with ACHA, none at all are seen with the use of SA and the levels observed using DHB are about 30% of those seen in ACHA. These lower levels of immonium ions in SA and DHB are also associated with much lower levels of backbone fragmentation compared to ACHA. Finally, a comparison of ACHA fragmentation was made using laser pulse length of 600 psec with the earlier studies employing 5 nsec pulse lengths. This study showed that the 600 psec pulse length gives rise to the onset of ionization at much lower levels of fluence and is associated with much higher levels of immonium ion formation than does the 5 nsec puls length. These observations are almost certainly the result of the dominance of the first mechanism described above, the direct desorption from the sample surface.